Preventive and therapeutic agent against liver disorder

ABSTRACT

A new preventive and therapeutic agent given by oral administration containing carnosine zinc salt as the effective component and, effective against alcoholic liver disorder, viral hepatitis or liver disorders caused by drugs, toxicants, radiation, etc.

TECHNICAL FIELD

The present invention relates to a new preventive and therapeutic agentagainst liver disorder (hepatopathy), which is particularly effectivefor the prevention and treatment against alcoholic liver disorder, viralhepatitis and liver disorders caused by drugs, toxic substances,radiation, etc.

BACKGROUND ART

The liver is the largest organ in human body and fulfills the mostimportant functions such as metabolism and storage of nutritionalsubstances, detoxicating various substances, etc. The liver maintainscirculatory dynamics under normal conditions as a circulatory systemnext to systemic circulation and pulmonary circulation. Therefore, whenhepatic cells are adversely affected either acutely or chronically bycauses such as toxic substances, drugs, alcohol, viruses, etc. passingthrough the liver or by malnutrition, radiation, cholestasia, etc., theentire body is adversely affected.

In liver disorder, characteristic changes are observed from clinical,biochemical and histological viewpoints.

Liver disorder is characterized by increase of hepatic enzymes such asglutamic pyruvic transaminase ("GPT"), glutamic oxaloacetic transaminase("GOT"), alkaline phosphatase ("AL-P"), sorbitol dehydrogenase ("SDH"),etc. in blood or by increase of serum bilirubin.

Measurements of activity of hepatic enzyme and bilirubin value in bloodare utilized for characterization and judgment of degrees of liverdisorder, and these methods are generally used for clinical examination.For example, increase of serum AL-P activity suggests physical blockingof extrahepatic bile duct, early development of liver cirrhosis orarrest of choleresis by drug. Increases of activity of serum GPT, GOTand SDH are common to all types of liver disorder and indicate damage ofhepatic cells (Merk Manual, 13th edition, Chapter 8, p. 836, 1977).

Liver disorder is associated with necrosis of hepatic cells, and thisnecrosis is histologically identifiable and provides excellent indicesto show degree of liver disorder.

Since the degree of liver disorder can be judged by conditions ofhepatic enzymes in blood, serum bilirubin and necrosis of hepatic cells,these indices are generally used in search of preventive and therapeuticagents against liver disorder. Various models of hepatitis have beendeveloped for experimental evaluation of hepatic disorder. Of thesemodels, the liver disorder caused by hepatic toxicants such asD-galactosamine, carbon tetrachloride, etc. are most akin to the viralhepatitis or the hepatitis caused by drug, toxicants, etc. as actuallyseen.

In the hepatic disorder caused by carbon tetrachloride, the linkage ofcarbon tetrachloride is severed in liver by cytochrome P-450, therebygenerating the highly toxic free radical (.CCl₃), and it is generallybelieved that this free radical causes the disorder by combining withthiol group of protein in hepatic cell membrane or by acceleratingperoxidation reaction of the lipids in cell membrane. (Biochem.pharmacol., Vol. 25, p. 2163, 1976, and Biochem. pharmacol., Vol. 21, p.49, 1972). As the result, it biochemically induces suppression ofprotein synthesis in liver and escape of hepatic enzymes such as GPT,GOT and SDH into blood. Histologically, it causes coagulation necrosis,edematous degeneration and fat formation, etc.

It is said that the models with disorder caused by carbon tetrachlorideand D-galactosamine are akin to the liver disorder due to chronicalcoholism. On the models with carbon tetrachloride, variousdescriptions have been given in literature: Amer. J. Path., Vol. 79, p.579, 1975; Virchowa Arch, B. Cell. Path., Vol. 26, p. 331, 1978; andSemirars in Liver Disease, Vol. 1, p. 143, 1981. It is described thatthis model is most akin to the liver disorder caused by viruses, drugs,toxicants, etc.

As liver disorders, there are acute hepatitis, chronic hepatitis, livercirrhosis, fatty liver, etc., and the causes are diverse such astoxicants, drugs, alcohol, viruses, malnutrition, radiation,cholestasia, etc. Various preventive and therapeutic agents have beenconceived against these liver disorders. Most of these drugs were thedrugs to prevent and treat the metabolic anomaly, which secondarilyoccurs when hepatic cells are affected, and they are disadvantageous inthat the therapeutic effects against liver disorders caused by specificcauses such as viruses are rather weak.

The present invention offers preventative and therapeutic agents againstvarious types of liver disorders.

DISCLOSURE OF THE INVENTION

The present inventors have found through experiments with model animalshaving hepatitis experimentally induced by carbon tetrachloride that,when carnosine zinc salt was given before liver disorder occurs, theincrease of hepatic enzymes in blood was effectively prevented, that itwas effective to prevent liver disorder before it occured, that the samepreventive effect was obtained when carnosine zinc salt was given afterliver disorder had actually occurred, and that carnosine zinc salt iseffective in prevention and treatment of liver disorder.

Specifically, the present invention relates to a preventive andtherapeutic agent against liver disorder, containing carnosine zinc saltas effective component, and the improvement of liver disorder bycarnosine zinc salt is revealed by significant decline in the increaseof GPT and GOT in blood and by the reduction of the degree of necrosisof hepatic cells.

Carnosine zinc salt of the present invention is a drug known for thetreatment of peptic ulcer, and its application and manufacturing methodare described in the Japanese provisional patent publication No.59-33270.

147 g of L-carnosine dissolved in 441 ml of pure water was added with asolution of 88.6 g of zinc chloride in 177 ml of pure water. 325 ml of4N sodium hydroxide aqeous solution was dropped into said reactionsolution while stirring up for 30 min., and the reaction was completed.After the reaction, the deposited precipitate was taken by filtrationand was rinsed well with water until the washing solution was turned toneutral. When dried at 40° C. for 2 days, it gave 175 g of colorlesspowder of L-carnosine zinc salt.

The analysis result of this substance is as follows:

    ______________________________________                                        Loss on drying (1 g; dried under reduced                                      pressure at 60° C. for hours)                                                                7.63%                                                   Zinc content (weight analysis)                                                                      23.20%                                                  Carnosine content (weight analysis)                                                                 76.81%                                                  Melting point         300° C. or more                                  I.R. spectrum (KBr, cm.sup.-1)                                                                      3280, 1620,                                                                   1480, 1385, 1260, 1120,                                                       1050, 1000, 980                                         ______________________________________                                    

PREVENTION OF EXPERIMENTAL LIVER DISORDER IN RAT EXPERIMENTAL METHOD

Carnosine zinc salt was suspended in 0.5% sodium carboxymethyl cellulose(CMC) solution to prepare 60 mg/ml and 20 mg/ml suspensions. This wasgiven to rats orally every day for 8 days at the rate of 0.5 ml/100 g sothat the dosage was to be 300 mg/kg and 100 mg/kg, respectively. To thecontrol group, the same volume of 0.5% CMC was given.

To prepare the model animals with liver disorder caused by carbontetrachloride, carbon tetrachloride mixed in olive oil to have a 10%(V/V) mixture was given orally at the rate of 4 ml/kg (0.4 ml/kg ascarbon tetrachloride) one hour after the final administration ofcarnosine zinc salt or 0.5% CMC. The animals were anesthetized byintraperitoneal injection of pentobarbital at 24 hours after carbontetrachloride was given, and blood was collected from carotid arteries.The blood was centrifuged at 3000 rpm for 15 minutes to fractionateserum, and GOT and GPT were determined (Reitman Frankel and Momosemodification).

From the isolated liver, the paraffined sections were prepared, stainedby H-E staining and were examined by histopathological examination.

For the experiment, male Wistar rats were used. With 10 rats in eachgroup, they were divided into three groups: the group given with carbontetrachloride (CCl₄) and 0.5% CMC only (CCl₄ +CMC), the group given withcarbon tetrachloride and 300 mg/kg or 100 mg/kg of carnosine zinc salt(CCl₄ +carnosine zinc salt) and the group given with 0.5% CMC only(CMC).

The results are as shown in the table below:

    ______________________________________                                                   Items of measurement                                               Specimen     GOT (KU)    GPT (KU)                                             ______________________________________                                        CCl.sub.4 + CMC                                                                            2115.9 ± 260.2                                                                         2243.8 ± 333.0                                    CCl.sub.4 + CAZ                                                               (100 mg/kg)  2167.7 ± 777.5                                                                         1917.7 ± 748.6                                    (300 mg/kg)   629.7 ± 121.2                                                                         427.7 ± 69.4                                      CMC          89.1 ± 3.2                                                                             42.5 ± 1.3                                        ______________________________________                                         (Note)-                                                                       CAZ represents carnosine zinc salt.                                           KU means KARMEN unit.                                                    

The result of this experiment suggests the effect of carnosine zinc saltto prevent the experimental liver disorder caused by carbontetrachloride.

In the acute toxicity test, female and male Wistar rats, each with bodyweight 150-200 g, were divided into the groups with 10 rats each, and 10g/kg each of carnosine zinc salt was given orally. By the observationfor 7 days, there was no case of death, showing very weak toxicity ofthis compound.

THE BEST WAY TO EXECUTE THE INVENTION

Adequate method to give carnosine zinc salt is oral administration.Dosage form may be any of suspensions, tablets, pills, capsules orpowder. Though dosage of carnosine zinc salt for prevention andtreatment of liver disorder may differ according to degree of liverdisorder, the effective daily dosage is 0.3 mg-30 mg/kg, preferably 1.5mg-15 mg/kg.

CAPABILITY OF EXPLOITATION IN INDUSTRY

As described above, carnosine zinc salt according to the presentinvention suppresses increase of GPT and GOT in blood, depending uponthe dosage. In the group with 300 mg/kg administration, it wassignificantly decreased at risk rate of 1%.

Also, in the histopathological observation, the suppression of necrosisof hepatic cells and fat formation were observed in the group given withcarnosine zinc salt.

Further, it was confirmed that acute toxicity of carnosine zinc salt ofthis invention is extremely weak.

Therefore, it is clear that carnosine zinc salt of this invention iseffective to the model animals with liver disorder caused by carbontetrachloride. Since these model animals with liver disorder due tocarbon tetrachloride are evaluated as good models with actual liverdisorder caused by viruses, drugs, toxicants, etc., it follows thereforethat carnosine zinc salt of this invention is very useful as thepreventive and therapeutic agent by oral administration against liverdisorders.

What is claimed is:
 1. The method of treating against liver disordercomprising administering orally to an individual, subject to liverdisorder, an agent containing carnosine zinc salt as an effectivecomponent and in an effective amount to treat said disorder.
 2. Themethod of claim 1 wherein said liver disorder is viral hepatitus.
 3. Themethod of claim 1 wherein said liver disorder is caused by drugs,toxicants or radiation.
 4. The method of claim 1 wherein said liverdisorder is caused by alcohol.